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The Impact on Receiving Waters of Pharmaceutical Residues and Antibiotic Resistant Faecal Bacteria Found in Urban Waste

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The Impact on Receiving Waters of Pharmaceutical Residues and Antibiotic Resistant Faecal Bacteria Found in Urban Waste The impact on receiving waters of pharmaceutical residues and antibiotic resistant faecal bacteria found in urban waste water effluents Thesis submitted to Middlesex University in partial fulfilment of the award of Doctor of Philosophy (Ph.D.) degree By Rebecca Tuckwell Supervisors: Prof. Mike Revitt, Prof. Hemda Garelick, Dr. Huw Jones Middlesex University, London, UK October, 2014 i Abstract Pharmaceuticals intended for human use are frequently detected in the aquatic environment. This is predominantly from their excretion following ingestion and subsequent discharge in domestic sewage. Wastewater treatment provides an opportunity to control their release to surface waters however, their removal is often incomplete. This thesis addresses this pharmaceutical pathway and the potential impact on the aquatic environment. The progress of bezafibrate, carbamazepine, ciprofloxacin and clarithromycin were monitored through the treatment stages (screened sewage, settled sewage and final effluent) of a large urban wastewater treatment plant (WWTP) and in surface waters up- stream and down-stream of the effluent discharge point. All except clarithromycin were detected in the screened sewage (369 – 2696 ng/L). Reductions in the pharmaceutical concentrations throughout the WWTP (22.5 – 94.3 %) indicate the removal of these compounds is variable. Bezafibrate and carbamazepine were observed at higher concentrations (67.5 - 305.5 ng/L) in surface water down-stream of the effluent discharge point compared to up-stream (31.0 – 116.7 ng/L). The presence of antibiotics in the environment may contribute to the dissemination of antibiotic resistance. The second part of this thesis monitors the prevalence of resistant faecal bacteria through WWTPs and in surface waters. Determination of antibiotic minimum inhibitory concentration (MIC) values for E.coli and E.faecium indicated that the WWTP did not influence the proportions of the resistant bacterial species. Elevated levels of E.coli with acquired ciprofloxacin resistance increased from not detectable in surface waters up-stream ii to 9.3% down-stream of the WWTP discharge point. The need for standardisation of the interpretation of MIC data is addressed. The potential of ciprofloxacin within surface water to select for ciprofloxacin resistant E.coli were investigated through microcosm studies in the third part of this study. A significant increase (p < 0.05) in the level of resistant E.coli was observed in microcosms exposed to ≥ 5 µg/L ciprofloxacin. At the ciprofloxacin levels typically detected in surface waters receiving treated effluent (<300 ng/L), the levels of resistance amongst E.coli were maintained. iii Contents Abstract ................................................................................................................................................ i Contents ......................................................................................................................................... iii List of figures ................................................................................................................................... x Abbreviations .............................................................................................................................. xviii Acknowledgments ........................................................................................................................ xix 1 Introduction .............................................................................................................. 1 1.1 Thesis objectives .......................................................................................................... 5 1.1.1 Rationale ......................................................................................................................................... 5 1.1.2 Aims ................................................................................................................................................ 6 1.2 Organisation of thesis .................................................................................................. 7 2 The Occurrence of Pharmaceuticals in Environmental Waters .................................. 10 2.1 Pharmaceuticals ........................................................................................................ 10 2.1.1 Pharmaceutical consumption ....................................................................................................... 10 2.2 Sources of pharmaceuticals into the aquatic environment ........................................... 11 2.2.1 Pharmaceuticals and the wastewater treatment process ............................................................ 12 2.3 Occurrence of pharmaceuticals in wastewater and surface water ............................... 18 2.4 Analytical methods to detect pharmaceuticals in environmental waters ...................... 20 iv 2.4.1 Sample preparation and analysis .................................................................................................. 20 2.4.2 Liquid chromatography-mass spectrometry (LC-MSn) .................................................................. 21 2.5 Legislation relating to the occurrence of pharmaceuticals in environmental waters ..... 23 2.5.1 The Water Framework Directive ................................................................................................... 24 2.5.2 Urban Wastewater Treatment Directive (UWWTD) ..................................................................... 24 2.5.3 Registration, evaluation, authorisation and restriction of chemicals (REACH) ............................. 25 2.5.4 European Medicines Agency (EMA) .............................................................................................. 25 3 Water quality indicator bacteria and antibiotic resistance ....................................... 28 3.1 Microbial indicators of water quality .......................................................................... 28 3.1.1 Coliform bacteria and Escherichia coli .......................................................................................... 29 3.1.2 Enterococci .................................................................................................................................... 30 3.1.3 Staphylococci ................................................................................................................................ 31 3.1.4 Pseudomonas aeruginosa ............................................................................................................. 32 3.2 Detection, enumeration and identification of indicator bacteria .................................. 32 3.2.1 Enumeration methods .................................................................................................................. 33 3.2.2 Identification of bacteria .............................................................................................................. 36 3.3 Antibiotic resistance in bacteria indicative of faecal contamination ............................. 40 3.3.1 Antibiotics and antibiotic action on bacteria ................................................................................ 40 v 3.3.2 Mechanisms of antibiotic resistance ............................................................................................. 43 3.3.3 Dissemination of antibiotic resistance .......................................................................................... 44 3.3.4 Antibiotic resistance in environmental waters.............................................................................. 47 3.3.5 Antibiotic resistance in E.coli ........................................................................................................ 48 3.3.6 Antibiotic resistance in enterococci .............................................................................................. 50 3.4 Antibiotic susceptibility testing ................................................................................... 53 3.4.1 Definition of resistance to antibiotics ........................................................................................... 55 4 Detection of Pharmaceuticals in the Urban Water Environment ............................... 58 4.1 Introduction ............................................................................................................... 58 4.1.1 Selection of pharmaceuticals ........................................................................................................ 59 4.2 Materials and methods for the analysis of pharmaceuticals in environmental waters .. 70 4.2.1 Chemicals and reagents ................................................................................................................ 70 4.2.2 Description of study area .............................................................................................................. 70 4.2.3 Sample collection .......................................................................................................................... 72 4.2.4 Water quality parameter analysis ................................................................................................ 75 4.2.5 Analytical method to determine target pharmaceutical concentrations...................................... 76 4.2.6 Validation of the analytical method to determine target pharmaceuticals in environmental waters 78 4.2.7 Prediction of pharmaceutical consumption .................................................................................
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